Automated molecular pathology apparatus having fixed slide platforms

ABSTRACT

Apparatus and methods for automatically staining or treating multiple tissue samples mounted on slides are provided, in which the slides and reagent bottles are held in fixed position, and the reagent, wash and coverslipping solutions brought to the slides. Alternatively, the slides are held in fixed position, while the reagent, wash and coverslipping solutions brought to the slides.

CROSS REFERENCE TO RELATED APPLICATION

This Application claims benefit of priority from U.S. ProvisionalApplication Ser. No. 60/375,679 filed Apr. 26, 2002.

FIELD OF THE INVENTION

The present invention is directed to apparatus for use in diagnosticmolecular pathology and, more particularly, to such apparatus used forthe automated staining and/or treating of tissue samples mounted onmicroscope slides.

BACKGROUND OF THE INVENTION

Molecular pathology is the examination at a molecular level of the DNA,mRNA, and proteins that cause or are otherwise associated with disease.From this examination important information about patient diagnosis,prognosis, and treatment options can be elucidated. The practice ofmolecular pathology is generally divided into two main areas: (i)analysis of DNA, mRNA, and proteins in intact cells (in-situ), and (ii)analysis of these biological materials after they have been extractedfrom tissues. The first category, to which the present invention isprimarily directed, has the advantage that it allows the pathologist orscientist to study the histopathologic architecture or morphology of thetissue specimen under the microscope at the same time that the nucleicacid or proteins are being assayed. These techniques includeimmunohistochemistry (IHC) which looks at proteins, in-situhybridization (ISH) which looks at nucleic acids, histochemistry (HC)which looks at carbohydrates, and enzyme histochemistry (EHC) whichlooks at enzyme chemistry. For example, ISH can be used to look for thepresence of a genetic abnormality or condition such as amplification ofcancer causing genes specifically in cells that, when viewed under amicroscope, morphologically appear to be malignant. ISH is also usefulin the diagnosis of infectious diseases as it allows detection not onlyof a microbial sequence but also of precisely which cells are infected.This may have important clinicopathologic implications and is aneffective means to rule out the possibility that positive hybridizationsignal may have come from an adjacent tissue of no clinical concern orfrom blood or outside contamination.

IHC utilizes antibodies which bind specifically with unique epitopespresent only in certain types of diseased cellular tissue. IHC requiresa series of treatment steps conducted on a tissue section or cells (e.g.blood or bone marrow) mounted on a glass slide to highlight by selectivestaining certain morphological indicators of disease states. Typicalsteps include pretreatment of the tissue section to remove the paraffinand reduce non-specific binding, retrieval of antigens or cellconditioning masked by cross-linking of the proteins from the chemicalfixatives, antibody treatment and incubation, enzyme-labeled secondaryantibody treatment and incubation, substrate reaction with the enzyme toproduce a fluorophore or chromophore highlighting areas of the tissuesection having epitopes binding with the antibody, counterstaining, andthe like. Most of these steps are separated by multiple rinse steps toremove unreacted residual reagent from the prior step. Incubations canbe conducted at elevated temperatures, usually around 37° C., and thetissue must be continuously protected from dehydration. ISH analysis,which relies upon the specific binding affinity of DNA or RNA probeswith unique or repetitive nucleotide sequences from the cells of tissuesamples or bodily fluids, requires a similar series of process stepswith many different reagents and is further complicated by varyingtemperature requirements.

In view of the large number of repetitive treatment steps needed forboth IHC and ISH, automated systems have been introduced to reduce humanlabor and the costs and error rate associated therewith, and tointroduce uniformity. Examples of automated systems that have beensuccessfully employed include the ES®, NexES®, DISCOVERY™, BENCHMARK™and Gen II® staining Systems available from Ventana Medical Systems(Tucson, Ariz.). These systems employ a microprocessor-controlled systemincluding a revolving carousel supporting radially positioned slides. Astepper motor rotates the slide carousel placing each slide under one ofa series of reagent dispensers positioned above the slides. As describedin U.S. Pat. No. 6,352,861 B1, bar codes on the slides and reagentdispensers fully automate the computer-controlled actuation of thedispensers onto the slides so that different reagent treatments can beperformed for each of the various tissue samples.

Instrumentation such as the Ventana Medical Systems ES®, Gen II® NexES®,BENCHMARK® and DISCOVERY® systems are fundamentally designed tosequentially apply reagents to tissue sections mounted on one by threeinch glass microscope slides under controlled environmental conditions.The instrument must perform several basic functions such as reagentapplication, washing (to remove a previously applied reagent), jetdraining (a technique to reduce the residual buffer volume on a slidesubsequent to washing), Liquid Coverslip™ application (a light oilapplication used to contain reagents and prevent evaporation), and otherinstrument functions.

The Ventana Medical Systems staining instruments mentioned above processslides on a rotating slide carousel. The instrumentation describedherein has the slides fixed in a stationary position and rotates thebasic processing stations above the fixed slides. The following detailsof how the slides are processed, the process algorithm, is the sameregardless of the physical configuration.

The process of staining tissue on a slide consists of the sequentialrepetition of the basic instrument functions described above.Essentially a reagent is applied to the tissue then incubated for aspecified time at a specific temperature. When the incubation time iscompleted the reagent is washed off the slide and the next reagent isapplied, incubated, and washed off, etc, until all of the reagents havebeen applied and the staining process is complete.

It is desirable to permit any staining protocol for any of the slidesbeing run, i.e. any combination of reagents and incubation times. Inaddition, to stain multiple slides as quickly as possible the instrumentshould process the slides simultaneously. This is feasible given thatmost of the time slides are just incubating, thus freeing up time toperform the washing, reagent application and other functions on otherslides.

One algorithm to accomplish simultaneous staining (sometimes referred toas the “random access” method) is to create a task and time schedule foreach slide in the run, then perform each task on each slide when theschedule calls for it. The problem with this method is that incubationtimes will not be accurate if the instrument is busy performing a taskon one slide when it is time to be washing another slide (therebycompleting incubation on that slide). The variation in incubation timeswill be unpredictable since the total number of slides and the slideprotocols vary.

Slide processing using a lock-step algorithm insures that all incubationtimes are accurate and predictable irrespective of the number of slidesprocessed or the variation in slide protocols. While incubation timesare assured, the lock step algorithm implies that incubation times mustbe an increment of the fundamental incubation time period. For example,with an incubation cycle of two minutes, the total incubation times mustbe multiples of two, i.e., two, four, six, eight etc. minutes induration. However, the preferred embodiment of the present inventionuses a four minute incubation time. Generally this is not a particularlimitation since typical incubation times are an order of magnitudelonger than the fundamental incubation period.

Prior art staining systems typically include either convection orradiation to warm the samples above laboratory ambient temperatures forsteps requiring elevated temperatures. Heating the slide improvesstaining quality by acceleration of the chemical reaction and can permita reaction temperature more closely matching body temperature (about 37°C.) at which antibodies are designed to react. While such convection orradiant heating systems have been generally suitable for IHC, which isantibody-based, they are less suitable for ISH, which is nucleicacid-based and requires higher and more precise temperature control inorder to denature DNA. In order to denature the DNA double helix of boththe target sample and the probe so as to render them single stranded,the temperature must be raised above the melting point of the duplex,usually about 94° C. Precise temperature control is also required in ISHto effect probe hybridization at the desired stringency. The selectedtemperature must be low enough to enable hybridization between probe andtarget, but high enough to prevent mismatched hybrids from forming.

Hot air convection, conduction or radiant heat heating units typicallyemployed with prior art automated tissue stainers do not permit thetemperature of individual slides to be separately controlled. With priorart systems all of the slides are heated to the same temperature at anygiven time during the process. For example, U.S. Pat. Nos. 5,645,114 and6,180,061 to Bogen et al. disclose a dispensing assembly adapted tocarry a plurality of microscope slides. Individual slide holderscontaining resistive heating units are provided. However, with theassembly taught by Bogen et al., all of the slides would be heated to acommon temperature because no means are disclosed for separate heatingcontrols or for shielding slides from heat generated by adjacent slides.

Other difficulties frequently encountered in both IHC and ISH testingresults from the manner in which the tissues are typically preserved.The mainstay of the diagnostic pathology laboratory has been for manydecades the formalin-fixed, paraffin-embedded block of tissue, sectionedand mounted upon glass slides. Fixation in such a preservative causescross-linking of macromolecules, both amino acids and nucleic acids.These cross-linked components must be removed in the case of ISH, toallow access of the probe to the target nucleic acid, and, in the caseof IHC, to allow the antibody to recognize the corresponding antigen.“Unmasking” the antigen and/or nucleic acid is typically accomplishedmanually with multiple pretreatment, protolytic digestion, and washsteps.

Prior to staining, complete removal of the paraffin is also required sothat it does not interfere with antibody or probe binding. Manualdeparaffinization normally is achieved by the use of two or threesuccessive clearing reagents that are paraffin solvents such as xylene,xylene substitutes or toluene. However, new automated methods that arelargely based on physical separation mechanisms are revealed in U.S.Pat. No. 6,544,798 B1 to Christensen et al., which do not require toxicsolvents and are aqueous-based.

The foregoing discussion of the prior art largely derives from Richardset al. U.S. Pat. No. 6,296,809, assigned to Ventana Medical Systems, inwhich there is described apparatus and methods for automaticallystaining or treating multiple tissue samples mounted on microscopeslides so that each sample can receive an individualized staining ortreatment protocol even when such protocols require differenttemperature parameters. More specifically, there is described in the'809 patent apparatus comprising a computer controlled, bar code driven,staining instrument that automatically applies chemical and biologicalreagents to tissue or cells mounted or affixed to standard glassmicroscope slides. According to the '809 patent, a plurality of slidesare mounted in a circular array on a carousel which rotates, as directedby the computer, to a dispensing location placing each slide under oneof a series of reagent dispensers on a second rotating carouselpositioned above the slides. Each slide receives the selected reagents(e.g. DNA probe) and is washed, mixed and/or heated in an optimumsequence and for the required period of time.

According to the '809 patent, individual slides are carried on thermalplatforms radially mounted to the carousel. Temperature sensors are alsomounted to the slide carousel, individually monitoring and controllingeach thermal platform separately. Apparatus made in accordance with the'809 patent is available commercially from Ventana Medical Systems, ofTucson, Ariz. as the DISCOVERY® or BENCHMARK® systems.

The present invention is a modification and improvement over the priorart including the apparatus and methods described in the '809 patent.More particularly, the present invention rather than bringing the slidesto the reagent, stain, and wash stations, brings the reagent, stain andwash stations to fixedly positioned slides. That is to say, in thepresent invention the slides are fixedly positioned in the apparatus,and the various washing, staining and reagent fluids are selectivelydelivered to the slides. Fixing the slides in position in the apparatussimplifies wiring to the heaters, and also eliminates the potential thata slide may be dislocated by rapid start and stop movement of the slidecarousel, which, in a worst case scenario could result in a domino ortrain-wreck effect where one dislocated slide hits the neighboring slidecausing that slide to dislocate, and so forth.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present invention will be seenfrom the following detailed description, taken in conjunction with theaccompanying drawings, wherein like numerals depict like parts, andwherein:

FIG. 1 is a perspective view of a first embodiment of an apparatus ofthe present invention shown with the slide cabinet shell removed, andFIG. 1A is an enlarged view showing details of portions of the reagenttransfer probes;

FIG. 2 is a perspective view of the apparatus of FIG. 1, firstembodiment shown in conjunction with a computer and other instrumentswith which it operates;

FIG. 3 is a perspective view and FIG. 3A is a partially exploded view ofdetails of the nozzle support portion of FIG. 1, first embodiment;

FIG. 4 is an exploded view of details of the slide plate portion of FIG.1, first embodiment;

FIGS. 5 and 6 are perspective views, from the top and the bottom,respectively, of portions of the slide plate portion of FIG. 1, firstembodiment;

FIG. 7 is a perspective view of the reagent support portion of FIG. 1,first embodiment;

FIG. 8 is a partially exploded perspective view showing two reagentbottles of FIG. 1, first embodiment;

FIG. 8A is a cross-sectional view of a reagent bottle, cap and insert,first embodiment;

FIG. 9 is a top plan view of two reagent bottles of FIG. 1, firstembodiment;

FIGS. 10A-10B are flow charts of the operation and control of FIG. 1,first embodiment;

FIG. 11 is a perspective view of an alternative embodiment and apparatusof the present invention shown with the slide cabinet shell removed;

FIG. 12 is a perspective view of the FIG. 11 alternative embodimentshown in conjunction with a computer and other instruments with which itoperates;

FIG. 13 is a perspective view showing details of the nozzle supportportion of the FIG. 11 alternative embodiment;

FIG. 14 is an exploded view of details of the FIG. 11 alternativeembodiment;

FIGS. 15 and 16 are perspective views, from the top and the bottom,respectively, of portions of the slide plate portion of the FIG. 11alternative embodiment;

FIGS. 17A and 17B are front and rear perspective views of the reagentdispenser and dispenser carrier portion of the FIG. 11 alternativeembodiment;

FIG. 18 is a perspective view showing details of the reagent dispenseractivator portion of the FIG. 11 alternative embodiment; and

FIGS. 19A and 19B are flow charts of the operation and control of theFIG. 11 alternative embodiment.

DETAILED DESCRIPTION OF THE INVENTION

Referring now in detail to the drawings wherein like parts aredesignated by like reference numerals throughout, there is illustratedin FIG. 1 a perspective view of the molecular pathology apparatusaccording to a first embodiment of the present invention which isdesignated generally by reference numeral 10. For the purposes ofclarity, several of the reagent bottles, as well as the cabinet shell,and liquid and air supply tubing and electrical wiring are omitted fromthe drawings. Apparatus 10 is designed to automatically stain orotherwise treat tissue mounted on microscope slides with nucleic acidprobes, antibodies, and/or other reagents in a desired sequence, timeand temperature. Tissue sections so stained or treated are then to beviewed under a microscope by a medical practitioner who reads the slidefor purposes of patient diagnosis, prognosis, or treatment selection.

In the first embodiment, apparatus 10 functions as one component ormodule of a system 12 (FIG. 2) which also comprises a host computer 14preferably a personal computer, monitor 16, keyboard 18, mouse 20, bulkfluid containers 22, waste container 23 and related equipment.Additional staining modules or other instruments may be added to system12 to form a network with computer 14 functioning as a server.Alternatively, some or all of these separate components could beincorporated into apparatus 10 making it a stand-alone instrument.

Referring also to FIGS. 3, 3A and 4-7, as set forth in greater detailbelow, a plurality of slide platforms 50 (FIGS. 4, 5) are mountedradially about a center point 32 of drawer 34 (FIG. 1) upon whichstandard glass slides 60 with tissue samples may be placed. Drawer 34 isslidably mounted in housing 30 on rails 40 or the like. The temperatureof each slide may be individually controlled by means of sensors and amicroprocessor, i.e. as taught in the above-mentioned '809 patent.

Each of the slide platforms 50 is connected through individual wires toa multiplexer (not shown) which is then in turn connected to amicroprocessor (not shown). A feature and advantage of the presentinvention which results from fixedly mounting the slide platforms indrawer 34 is that each of the heaters and thermal sensors may behardwired thereby eliminating the need for a slip ring assembly or rotorcouplings, as well as complex stepping motors, etc. for locating andpositioning a rotating slide carousel as required in prior art devices.Also, the possibility that a slide or slides may be shifted ordislocated during rapid start and stop rotation of the slide carousel iseliminated.

In one embodiment, a plurality of slots or channels are formed on thetop surface of each of the slide heaters, i.e. the interface surfacebetween the slide heater and the slide, for gathering and venting gasbubbles as may form during heating, i.e. in accordance with co-pendingU.S. application Ser. No. 09/953,417, filed Sep. 11, 2001, and assignedto the common assignee, which disclosure is incorporated herein byreference.

Referring also to FIGS. 1, 2, 5 and 6, drawer 34 supports a slidesupport tray 33 which in turn supports a circular pan 35 having aperipherial wall 36 serving as a splash guard, a peripheral trough 37and a central drain 38, i.e. at center point 32, both connected to drainlines 39 which in turn are connected to waste container 23. Drawer 34 isslidably mounted in housing 30 on rails 40. Rails 40, in a preferredembodiment, comprise three piece telescoping rails so that the drawer 34may be slid clear of housing 30 to permit access to all of the slideplatforms 50 for slide loading and removal. A damping means such as apneumatic means, electromotive means, mechanical spring damper or thelike preferably is provided to smooth movement of the drawer whereby toavoid possible dislodging of slides, particularly when the drawer isclosed. Slide support tray 33 is supported on a lift mechanism such aspneumatic cylinders 52 (see FIG. 1), which automatically index to permitthe slide support tray 33 to move up and down so that the slide supporttray 33 and circular pan 35 may be dropped to permit wall 36 to clearthe nozzle support 100 when the drawer is slid in and out of theapparatus.

Slide drawer 34 is divided into thirty-five equal pie-shaped sections70. Thirty of the pie-shaped sections 70 are occupied by slide platforms50 while the five remaining pie-shaped sections 70A (FIG. 4) at the rearof the drawer are devoid of slide platforms 50. In other words, a row ofthirty slide platforms 50 are radially mounted on drawer 34 and evenlyspaced from one another, except at the ends of the row.

However, the invention is not limited to thirty active slide locations,and more or fewer slide locations may be employed. An alternativeembodiment may be implemented by aligning the platforms 50 linearly,which results in potentially limitless number of platforms.

Referring to FIGS. 1 and 3, a nozzle support 100 is concentrically androtatably mounted above slide drawer 34. Nozzle support 100 is mountedon a shaft (not shown) supported by a bridge 110, and driven by acomputer controlled stepping motor and drive belt (not shown), androtates plus or minus approximately 180° from a home position 104 at therear of the drawer. The computer controlled stepper motor and drive beltare conventional in this art. Accordingly, details are omitted for thesake of clarity.

Nozzle support 100 carries the various slide treatment stations, otherthan the reagent dispensing location. Thus, nozzle support 100 carriesdual rinse nozzle block 102, volume adjust/stringency block 103, LiquidCoverslip™ evaporation inhibitor liquid application block 105, vortexmixer air jet block 106, jet drain 108, and the like, all for preparinga slide for staining, stain removal, and the like, and to clear barcodes (not shown) carried on the slides, and a bar code reader 109, allas described in detail in U.S. Pat. Nos. 5,595,707; 5,650,327;5,654,199; or 5,654,200 to Copeland et al, which disclosures areincorporated herein by reference. In other words, nozzle support 100carries all of the functions for slide preparation, cleaning, reagentmixing, Liquid Coverslip™ application, etc. other than reagentapplication, as described, e.g. in the '707 patent to Copeland et al.,plus wash stations 121, 122 for the reagent application probes as willbe described in detail below.

Preferably, but not necessarily, the various rinse nozzle blocks, vortexmixer air jet blocks, jet drain, etc. are arranged adjacent to oneanother so that the nozzle support 100 may be indexed and advanced in a“lock-step” manner to sequentially treat a slide according to anaccepted protocol. For example, jet drain 108 may be arrangedimmediately adjacent rinse nozzle blocks 102 so that nozzle support 100may be advanced in “lock step” manner past a selected slide, and theslide rinsed and fluid stripped, etc. Also, if desired, vortex mixer airjet blocks 106 may be oriented to impinge simultaneously on two adjacentslides.

For the sake of clarity, fluid and air supply tubing for the severalslide treatment stations have been omitted from the drawings. It will beunderstood, however, that the fluid and air supply tubing are made longenough to permit the valve plate to rotate plus or minus approximately190° from a home position at the rear of the apparatus so that eachslide treatment station can reach each slide 60. A pair of wash stations121, 122 spaced two thirty-fifths of a revolution (approximately 20°)apart as will be described in detail hereinafter, are also attached toand radially extend beyond the periphery of the nozzle support 100, androtate with the nozzle support 100.

Reagent support 300 is fixedly mounted to bridge 110 vertically abovenozzle support 100, which arch in turn is fixedly mounted within housing30. A plurality of reagent bottles 302 are removably mounted withinrecesses 304 formed equally spaced adjacent the periphery of reagentsupport 300. In the illustrated embodiment, a total of thirty-fivereagent bottles are mounted on the reagent support 300, spacedapproximately one thirty-fifth (approximately 10°) apart.

The reagents may include any chemical or biological materialconventionally applied to slides including nucleic acid probes orprimers, polymerase, primary and secondary antibodies, digestionenzymes, pre-fixatives, post-fixatives, readout chemistry,counterstains, and the like.

Referring also to FIG. 8, the reagent bottles 302 each comprise acylindrical hollow body 305 closed at the bottom end by an integrallyformed bottom wall. Preferably, the inside bottom wall of each bottle iscone shaped, i.e. as shown in phantom at 303 to facilitate reagentpick-up by reagent transfer probes as will be described in detail below.Each bottle 302 includes an integrally formed bracket 306 which servesto maintain the bottles 304 at a desired height in reagent support 300,and which may serve also to permit the stringing together of a pluralityof like bottles 302. Accordingly, each bracket 306 includes a hingeelement 308 for cooperating with a hinge element 310 of an adjacentbottle 302. In the illustrated embodiment, hinge elements 308 and 310are shown as conventional pin-hinges in which the upper hinge 310includes a pin 312 which fits into the lower hinge 308, i.e. similar toa conventional door hinge. However, bottles 302 may be hinged togetherin a variety of ways.

Bracket 306 preferably includes a flat surface 314 upon which is carrieda bar code 316 for identifying the contents of the bottle 302. Bottles302 also include an insert 318 having a tapered top surface 320 andinlet 319 fitted in the top end of the bottles for locating a reagenttransfer probe 328 as will be described in detail hereinafter, and a cap322 which may be either twist or snap-fitted to the bottle 302 forsealing the bottle 302.

Probe 328 is tapered at a lower end to fit closely in tapered conicalbottom 303 of bottle 302. A vent 321 is formed in insert 318 forequalizing air pressure as reagent is removed. Cap 322 includes atapered inner surface and plug 323 for sealing inlet 319.

Making brackets 306 attachable to one another permits one to assemble achain of reagents for use, and also to remove the chain of reagents sothat the reagents may be refrigerated, for example, overnight when notin use.

Referring next to FIGS. 7, 8 and 9, the side walls 324 of brackets 306are tapered so that a pie-shaped space 326 is formed between two bottleswhen two bottles are fastened together in a string, and mounted inrecesses 304 in the reagent support 300, thereby exposing holes 328formed through reagent support 300. Holes 328 are formed in the sameconcentric circle as recesses 304, and are spaced equidistant betweenadjacent recesses 304. The purpose of pie-shaped spaces 326 and holes328 is to provide clearance for reagent transfer probes 402, 404 as willbe described in detail below.

Referring again to FIG. 1, an arm 400 is rotatably mounted on arch 405concentrically above reagent support 300, and carries a pair of reagenttransfer probes 402, 404 located at the distal end of arm 400 and spacedapproximately 10° apart. Arm 400 also carries a bar code reader 406 forreading bar codes 316 on the reagent bottles. Arm 400 is rotatablydriven by a computer driven stepping motor (not shown), and rotates plusor minus approximately 185° in either direction from a home position410.

Reagent transfer probes 402 and 404, which are identical to one another,preferably comprise automatic pipette metering/dispensing pick-updevices designed to aspirate or “sip” reagent from a reagent bottle,move to a slide, and then “spit” or deposit the reagent onto the slide.“Sip” and “spit” automatic pipette/metering dispensing pick-up devicesare described in published PCT Application No. PCT/US99/04379, whichdisclosure is incorporated herein by reference. Reagent transfer probes402 and 404 are carried on the distal end of arm 400 and are spaced fromone another so that when one of the probes, e.g. probe 402 is locatedcentrally over a slide 60, the other reagent transfer probe 404 may becentrally positioned over one of the two probe wash stations 121 or 122.Pneumatic cylinders 403 a, 403 b selectively raise and lower probes 402and 404 into one of the following positions: a raised transport positionabove the tops of the bottles 302 where the arm 400 is free to rotate; areagent drawing position wherein one of the probes is inserted into aselected reagent bottle 302 wherein a measured amount of reagent may bedrawn into the probe; a reagent dispensing position wherein a reagenttransfer probe containing reagent is disposed in the pie-shaped space326 between two reagent bottles, above a selected slide to dispensereagent thereon; and a cleaning position wherein the other probe, i.e.the probe not being used to dispense reagent, is operatively disposed inone of probe washing stations 121 or 122 which straddle the slide beingdispensed upon. While the apparatus of the present invention could bemade with only a single reagent transfer probe, providing two spacedreagent transfer probes improves cycle speed since reagent metering maybe accomplished using one of the two reagent transfer probes while theother of the two reagent transfer probes is going through the wash cycleas will be described below. That is to say, while one of the reagenttransfer probes, e.g. reagent transfer probe 402 is dispensing reagentonto a slide, the other reagent transfer probe, i.e. idle reagenttransfer probe 404 may be lowered to a probe wash station 121 where theidle reagent transfer probe may be rinsed inside and out at the sametime.

Referring to FIGS. 10A and 10B, the overall process is as follows:

A plurality of specimen-bearing slides 60 are mounted on the slideplatforms 50, selected reagent bottles 302 mounted in the reagentsupport 300, the slide drawer is closed and the slide and reagent barcodes are read. The computer calculates the master protocol and thendownloads the run steps for the entire run, the nozzle support 100 isindexed to the first slide, and the slide is washed and prepared forstaining or other treatment in accordance with the pre-programmed runsteps by advancing the nozzle support 100 in “lock-step” manner. In themeanwhile, probe arm 400 is rotated to the appropriate reagent bottle302, one of the two reagent transfer probes 402 or 404 is indexed overthe selected reagent bottle, and the probe lowered to aspirate ameasured amount of the desired reagent. The reagent-containing transferprobe is then raised, and the arm 400 moved to the selected slide wherethe loaded reagent transfer probe is lowered to just over the slide, andthe reagent dispensed on the slide. In the meanwhile, the idle reagenttransfer probe is lowered into one of the washing stations 121 or 122,wherein the reagent transfer probe is washed inside and out. Bothreagent transfer probes 402 and 404 are then raised, and the processrepeated, but using the reagent transfer probe just cleaned in theprevious step to aspirate and dispense reagent onto the next slide. Asbefore, simultaneously with dispensing the reagent onto the slide as inthe previous case, the idle reagent transfer probe is washed while theactive reagent transfer probe is dispensing reagent onto the new slide.

The foregoing steps are repeated until all of the slides are processed.For convenience, in the illustrated embodiment, the dwell time at eachslide station is six and two-thirds seconds. This comes from dividing afour minute cycle time into thirty-six time spaces, one time space foreach of the thirty slide positions plus five blank slide positions forovertravel of the slide support, plus one “virtual” slide position forreturning the nozzle support 100 from the last slide position to thefirst slide position. The virtual slide position allows the nozzlesupport 100 to return to the other end of its travel range in anuninterrupted fashion.

The staining algorithm used on the aforesaid Ventana systems avoidsscheduling problems associated with random access methods by using a“lock step” method. The lock step algorithm requires that the nozzlesupport 100 which holds the processing functions be rotated one slideposition index every “n” seconds, termed the slide index time. The slideindex time is preferably as short as possible but long enough that thefunction that requires the longest time can be completed within theindex time. In the embodiments of the inventions herein, “n” is six andtwo-thirds seconds. Index times are usually on the order of severalseconds. The time for one complete rotation of the nozzle support 100,termed the fundamental incubation period, will then be “n” times thenumber of slide positions, including blank and virtual slide positions.(For example, if the slide index time is six seconds and there aretwenty slide positions, the incubation time period will be 120 secondsor two minutes.)

Throughout the entire run the nozzle support 100 is indexed one slideposition every “n” seconds. After the index, the system checks theschedule to see if any of the slides at each of the processing stationsrequire the function of that station. For example, if the slide at thewashing station is scheduled for washing, that slide is washed.Similarly if the slide at the reagent application station is scheduledfor the application of a new reagent, then the new reagent is applied.

The above-described invention has several advantages over the prior art.For one, making the slide plate fixed in position eliminates thepossibility of a slide being dislocated (and the sample dislodged)during the rapid start-stop rotational movement of a conventionalrotating slide carousel. Also, employing two transfer syringes insuresbetter cleaning of transfer syringes without increasing cycle time.

Also, since none of the moving elements, i.e. nozzle support 100 andprobe support arm 400 need travel more than approximately 190° in eitherdirection, all electrical connections, and air and fluid connections canbe achieved without the need for slip ring or rotary connections, sincethe hoses and wires are quite capable of taking twistings of 190° plus.

The instrumentation described herein may or may not have the ability tocontinuously rotate the nozzle support. The nozzle support 100 may needto return to a starting position before rotation has exceeded 360degrees. This may also be required when the slides are rotated on acarousel and the processing functions are fixed above the slides.Similarly, other non-rotating designs are possible such as linear or twodimensional configurations. In these cases there will be a requirementto move the slides or processing functions back to the original startingposition during the staining run. In most cases it is likely that thetime required to do this will exceed the index time which violates thefundamental requirement of the lock step algorithm. The lock stepalgorithm can still be utilized through the concept of the “virtualslide” previously mentioned. The virtual slide is added to the totalnumber of actual slide positions so that the index time period assignedto the virtual slide may be used to move the slides or processingstations back to the starting position. Thus accurate and predictableincubation times are maintained.

While one embodiment of the invention has been described, the inventionis susceptible to modification. For example, instead of using one or apair of transfer syringes on an overhead arm, the reagent carousel couldcarry a plurality of micro-delivery reagent fluid dispensers such asdescribed in U.S. Pat. Nos. 6,045,759; 6,416,713; or 6,192,945.Moreover, while the use of individually heated thermal platforms ispreferred, the slides may be heated using conventional heatingtechniques.

Referring to FIGS. 11-19, there is illustrated a second embodiment ofthe present invention. FIG. 11 is a perspective view of a molecularpathology apparatus according to the second embodiment which isdesignated generally by reference numeral 1000. For the purposes ofclarity, all but one of the reagent dispensers and dispenser carriers,as well as the cabinet shell, and liquid and air supply tubing andelectrical wiring are omitted from the drawings. Apparatus 1000 isdesigned to automatically stain or otherwise treat tissue mounted onmicroscope slides with nucleic acid probes, antibodies, and/or otherreagents in a desired sequence, time and temperature. Tissue sections sostained or treated are then to be viewed under a microscope by a medicalpractitioner who reads the slide for purposes of patient diagnosis,prognosis, or treatment selection.

In one embodiment, apparatus 1000 (FIG. 12) functions as one componentor module of a system which also comprises a host computer 1014preferably a personal computer, bulk fluid containers 1022, wastecontainer (not shown) and related equipment. Additional staining modulesor other instruments may be added to the system to form a network withcomputer 1014 functioning as a server. Alternatively, some or all ofthese separate components could be incorporated into apparatus 1000making it a stand-alone instrument.

Referring also to FIGS. 14, 15 and 18, as set forth in greater detailbelow, a plurality of slide platforms 1050 are mounted radially about acenter point 1032 of slide support assembly 1099 upon which standardmicroscope glass slides 1060 with tissue samples may be placed. Drawer1034 is slidably mounted in housing 1030 on rails 1040 or the like. Thetemperature of each slide may be individually controlled by means oftemperature sensors and a microprocessor, i.e. as taught in theabove-mentioned '809 patent.

As in the case of the first embodiment, each of the slide platforms 1050is connected through individual wires to a multiplexer (not shown) whichis then in turn connected to a microprocessor (not shown). Also, as inthe case of the first embodiment, a plurality of slots or channels areformed on the top surface of each of the slide heaters, i.e. theinterface surface between the slide heater and the slide, for gatheringand venting gas bubbles as may form during heating, i.e. in accordancewith co-pending U.S. application Ser. No. 09/953,417, filed Sep. 11,2001, and assigned to the common assignee, which disclosure isincorporated herein by reference.

Referring to FIGS. 14-16, drawer 1034 supports a slide support assembly1099 which is comprised of a slide support 1033 which in turn supports acircular pan 1035 having a peripheral wall 1036 serving as a splashguard, a peripheral trough 1037 and a central drain 1038, i.e. at centerpoint 1032, both connected to drain lines 1039 which in turn areconnected to waste container 1023. Drawer 1034 is slidably mounted inhousing 1030 on rails 1040. Rails 1040, in a preferred embodiment,comprise three-piece telescoping rails so that the drawer 1034 may beslid clear of housing 1030 to permit access to all of the slideplatforms 1050 for slide loading and removal. A damping means such as apneumatic means, electromotive means, mechanical spring damper or thelike (not shown) preferably is provided to smooth movement of the drawerwhereby to avoid possible dislodging of slides, particularly when thedrawer is closed. Slide support assembly 1099 is supported on a liftmechanism such as pneumatic cylinders 1052 (see FIG. 11), whichautomatically index to permit the slide support assembly 1099 to move upand down so that the slide support plate 1033 and circular pan 1035 maybe dropped to permit wall 1036 to clear the nozzle support 1100 when thedrawer is slid in and out of the apparatus.

Slide support assembly 1099 is allocated into thirty-five equalpie-shaped sections 1070. Thirty of the pie-shaped sections 1070 areoccupied by slide platforms 1050 while the five remaining pie-shapedsections 1070A (FIG. 15) at the rear of the drawer are devoid of slideplatforms 1050. In other words, a row of thirty slide platforms 1050 areradially mounted within slide support assembly 1099 and evenly spacedfrom one another, except at the ends of the row.

However, the invention is not limited to thirty active slide locations,and more or fewer slide locations may be employed. An alternativeembodiment may be implemented by aligning the platforms 1050 linearly,which results in potentially limitless number of platforms.

Referring to FIGS. 11, 14 and 18, a nozzle support 1100 isconcentrically and rotatably mounted above slide support assembly 1099.Nozzle support 1100 is mounted on a shaft 1113 which in turn isrotatably mounted on a bridge 1112, and driven by a computer controlledstepping motor and drive belt (not shown), and rotates plus or minusapproximately 190° from a home position at the rear of the drawer. Thecomputer controlled stepper motor and drive belt are conventional inthis art. Accordingly, details are omitted for the sake of clarity.

With respect to FIG. 13, nozzle support 1100 carries the various slidetreatment stations, other than the reagent dispensing station. Thus,nozzle support 1100 carries dual rinse nozzle block 1102, volumeadjust/stringency block 1103, Liquid Coverslip™ evaporation inhibitorliquid application block 1105, vortex mixer air jet block 1106, jetdrain 1108, and the like, all for preparing a slide for staining, stainreagent removal, and the like, and to clear the bar codes (not shown)carried on the slides, and a bar code reader 1109, all as described indetail in U.S. Pat. No. 5,595,707 to Copeland et al, which disclosure isincorporated herein by reference. In other words, nozzle support 1100carries all of the functions for slide preparation, cleaning, reagentmixing, Liquid Coverslip™ application, etc. other than reagentapplication, as described in the '707 patent to Copeland et al.

Preferably, but not necessarily, the various rinse nozzle blocks, vortexmixer air jet blocks, jet drain, etc. are arranged adjacent to oneanother so that the nozzle support 1100 may be indexed and advanced in a“lock-step” manner to sequentially treat a selected slide according toan accepted protocol. For example, jet drain 1108 may be arrangedimmediately adjacent rinse nozzle blocks 1106 so that nozzle support1100 may be advanced in “lock step” manner past a selected slide, andthe slide rinsed and fluid stripped, etc. Also, if desired, vortex mixerair jet blocks 1106 may be oriented to impinge simultaneously on twoadjacent slides.

For the sake of clarity, fluid and air supply tubing for the severalslide treatment stations have been omitted from the drawings. It will beunderstood, however, that the fluid and air supply tubing are made longenough to permit the valve plate to rotate plus or minus approximately190° from a home position at the rear of the apparatus so that eachslide treatment station can reach each slide 1060.

With respect to FIGS. 11 and 14, reagent support 1300 is fixedly mountedto a cage 1110 vertically above nozzle support 1100, which cage in turnis rotatably mounted for rotation on a shaft 1113, which in turn isdriven by a stepper motor and belt (not shown). A plurality of reagentdispensers 1302 are removably mounted equally spaced adjacent theperiphery of reagent support 1300. In the illustrated embodiment, atotal of thirty-five reagent dispensers are carried by the reagentsupport 1300, spaced approximately one thirty-fifth (approximately 10°)apart. Preferably, the reagent dispensers are mounted on curveddispenser carriers 1303, which in turn are mounted on reagent support1300 (see FIG. 17A).

Curved dispenser carrier 1303 permits a lab worker to assemble a kit ofreagent dispensers for use, and also to remove the kit of reagentdispensers so that the reagents may be refrigerated, for example,overnight when not in use.

The reagents may include any chemical or biological materialconventionally applied to slides including nucleic acid probes orprimers, polymerase, primary and secondary antibodies, digestionenzymes, pre-fixatives, post-fixatives, readout chemistry,counterstains, and the like.

Referring in particular to FIGS. 17A and 17B, the reagent dispensers1302, are shown mounted on dispenser carrier 1303.

Referring to both FIGS. 17A and 17B, each of the dispensers 1302 carriesa plate 1307 upon which may be mounted a bar code which may be read by abar code reader (not shown).

Referring also to FIG. 18, a hammer or piston 1400 is carried by an arm1402 which is mounted on shaft 1113 for rotation with the shaft. Hammer1400 is keyed to move with the nozzle support 1100, and is verticallyand concentrically operatively aligned over the reagent dispensers 1302.Hammer or piston 1400 comprises a servo or piston 1404 for movingvertically into engagement with the top of a selected reagent dispenser,and force a metered quantity of reagent from the dispenser i.e. asexplained in detail in U.S. Pat. No. 6,192,945. Thus, in order todispense a selected reagent on a selected slide, the nozzle support 1100is rotatably moved to a dispense position vertically over the selectedslide which had been previously prepared, e.g. washed, etc., by thetreatment stations carried on the nozzle support 1100. Since the hammeris keyed to move with the nozzle support 1100, there is no time wastedin moving the hammer or piston, and the hammer or piston is verticallyaligned. A particular feature and advantage of the present invention isthat the reagent support 1300 requires neither electrical nor plumbingconnections. Thus, the reagent support 1300 is free to rotate in anydirection, without limitation.

Referring to FIGS. 19A and 19B, the overall process is as follows:

A plurality of specimen-bearing slides 1060 are mounted on the slideplatforms 1050, and selected reagent dispensers 1302 are mounted on thereagent support 1300. The slide drawer is closed and the slide andreagent bar codes are read. The computer calculates the master protocoland then downloads the run steps for the entire run, the nozzle support1100 and hammer or piston 1400 are indexed to the first slide, and theslide is washed and prepared for staining or other treatment inaccordance with the pre-programmed run steps by advancing the nozzlesupport 1100 in “lock-step” manner. In the meanwhile, reagent support1300 is rotated to locate a selected reagent dispenser 1302 over theselected slide 1060, and the hammer or piston 1400 activated to dispensea measured amount of the desired reagent. The process is repeated for asecond selected slide, and so forth.

Still other changes may be made without departing from the spirit andscope of the invention.

1. An automatic slide processing apparatus comprising: (a) a slidesupport; (b) a reagent support; (c) a nozzle support; and (d) a reagentdispenser, wherein the slide support and the reagent support areoperatively fixedly positioned, and the nozzle support is selectivelyrotatably movable relative to the slide support and the reagent support.2. An apparatus as claimed in claim 1, wherein the slide support carriesa plurality of spaced slide support platforms.
 3. An apparatus accordingto claim 2, wherein said slide support platforms include heaters and/ortemperature sensors.
 4. An apparatus as claimed in claim 1, wherein saidslide support includes at least one liquid drain.
 5. An apparatusaccording to claim 1, wherein said slide support is slidably mounted inthe apparatus to permit loading and unloading of slides in the slidesupport.
 6. An apparatus according to claim 5, wherein said slidesupport is slidably mounted on rails.
 7. An apparatus according to claim6, and further including a damper for damping smoothing movement of therails.
 8. An apparatus according to claim 1, wherein said slide supportis vertically adjustably supported on a lift mechanism.
 9. An apparatusaccording to claim 1, wherein said reagent dispenser is selectivelymoveable relative to the reagent support.
 10. An apparatus according toclaim 9, wherein said reagent dispenser comprises robotic dispensingpipettes.
 11. An apparatus according to claim 10, wherein said roboticdispensing pipettes are vertically moveable between an operatingposition and a transport position.
 12. An apparatus according to claim9, wherein said reagent dispenser comprises two spaced roboticdispensing pipettes.
 13. An apparatus according to claim 1, wherein thenozzle support carries at least one vortex air jet or jet drain.
 14. Anapparatus according to claim 1, wherein the nozzle support carries atleast one applicator for applying a cover fluid to the slide.
 15. Anapparatus according to claim 1, wherein the nozzle support is rotatablymoveable plus or minus approximately 180° from a home position.
 16. Anapparatus according to claim 9, wherein said reagent dispenser isrotatably moveable plus or minus approximately 185° from a homeposition.
 17. An apparatus according to claim 1, and further comprisinga slide bar code reader carried on the nozzle support and/or carriedwith the reagent dispenser.
 18. An apparatus according to claim 1, andfurther comprising a processor for controlling movement of the nozzlesupport, and the reagent dispenser.
 19. An apparatus according to claim1, and further comprising a dispenser wash station mounted on the nozzlesupport.
 20. An apparatus according to claim 19, and comprising twodispenser wash stations mounted spaced from one another on the nozzlesupport.
 21. (canceled)
 22. A method for processing a plurality ofslides each having biological samples thereon comprising the steps of:(a) preparing a first slide for biological treatment with a reagent; (b)moving a reagent dispenser to a selected reagent container; (c) drawinga pre-selected volume of reagent from the reagent container into thereagent dispenser; (d) moving the reagent dispenser to the first slide;(e) dispensing said pre-selected volume of reagent from the reagentdispenser onto the first slide; (f) preparing a second slide fortreatment with a reagent; (g) cleaning the reagent dispenser; (h) movinga reagent dispenser to a selected reagent which may be the same ordifferent from the reagent of step (b); (i) drawing a pre-selectedamount of reagent from the reagent container selected in step (h) intothe reagent dispenser; (j) moving the reagent dispenser to said secondslide; (k) dispensing said pre-selected volume of reagent selected instep (h) from the reagent dispenser onto said second slide; (l) cleaningthe reagent dispenser; and (m) repeating steps (h)-(l).
 23. The methodof claim 22, wherein two reagent dispensers are provided, and includingthe step of cleaning one of the reagent dispensers while the otherreagent dispenser is employed to draw and dispense reagent onto aselected slide.
 24. A method according to claim 22, wherein said reagentdispenser comprises a robotic syringe, and reagent is taken up byaspiration.
 25. An automatic slide processing apparatus comprising: (a)a slide support; (b) a reagent support; (c) a nozzle support; and (d) areagent dispenser, wherein the slide support is operatively fixedlypositioned, and the nozzle support and reagent support are selectivelyindependently movable relative to the slide support.
 26. An apparatus asclaimed in claim 25, wherein the slide support carries a plurality ofspaced slide support platforms.
 27. An apparatus according to claim 26,wherein said slide support platforms include heaters and/or temperaturesensors.
 28. An apparatus as claimed in claim 25, wherein said slidesupport includes at least one liquid drain.
 29. An apparatus accordingto claim 25, wherein said slide support is slidably mounted in theapparatus to permit loading and unloading of slides in said slidesupport.
 30. An apparatus according to claim 29, wherein said slidesupport is slidably mounted on rails.
 31. An apparatus according toclaim 30, and further including a damper for damping smoothing movementof the rails.
 32. An apparatus according to claim 30, wherein said slidesupport is vertically adaptably supported on a lift mechanism.
 33. Anapparatus according to claim 29, wherein a reagent dispenser activatoris selectively rotatably moveable relative to the reagent support. 34.An apparatus according to claim 33, wherein said reagent dispenseractivator is fixed to move in synchronization with said nozzle support.35. An apparatus according to claim 34, wherein said reagent dispenseractivator comprises a hammer or piston.
 36. An apparatus according toclaim 35, wherein the nozzle support carries at least one vortex air jetand/or jet drain.
 37. An apparatus according to claim 25, wherein thenozzle support carries at least one applicator for applying a coverfluid to the slide.
 38. An apparatus according to claim 25, wherein thenozzle support is rotatably moveable plus or minus approximately 180°from a home position.
 39. An apparatus according to claim 25, whereinsaid reagent support is rotatably moveable in either direction from ahome position.
 40. An apparatus according to claim 25, and furthercomprising a slide bar code reader carried on the nozzle support.
 41. Anapparatus according to claim 25, and further comprising a reagent barcode reader operatively positioned relative to said reagent support. 42.An apparatus according to claim 25, and further comprising a processorfor controlling movement of the nozzle support and reagent dispenser.43. A method for processing a plurality of slides each having biologicalsamples thereon comprising the steps of: (a) preparing a first fixedlypositioned slide for biological treatment with a reagent; (b) moving aselected reagent dispenser to the first slide; (c) dispensing apre-selected volume of reagent from the reagent dispenser onto the firstslide; (d) preparing a second fixedly positioned slide for treatmentwith a reagent; (e) moving a selected reagent dispenser which may be thesame or different from the reagent dispenser of step (b) to the secondslide; (f) dispensing a pre-selected volume of reagent from the reagentdispenser onto said second slide; and (g) repeating steps (d)-(f).
 44. Amethod according to claim 43, wherein a reagent dispenser activator ismoved independently of said reagent dispensers.
 45. An automatic slideprocessing apparatus comprising: (a) a slide support; (b) heatingelements for heating the slides; (c) a nozzle support having nozzles forapplying liquids, said nozzle support and said slide support movingrelative to one another such that the nozzles may apply liquids to theslides; (d) a reagent support; and (e) a reagent dispenser wherein saidreagent dispenser is operative to directly access the slide support. 46.An apparatus as claimed in claim 45, wherein the slide support carries aplurality of spaced slide support platforms.
 47. An apparatus as claimedin claim 45, wherein said slide support includes at least one liquiddrain.
 48. An apparatus according to claim 45, wherein said slidesupport is slidably mounted in the apparatus to permit loading andunloading of slides in the slide support.
 49. An apparatus according toclaim 48, wherein said slide support is slidably mounted on rails. 50.An apparatus according to claim 49, and further including a damper fordamping smoothing movement of the rails.
 51. An apparatus according toclaim 45, wherein said slide support is vertically adjustably supportedon a lift mechanism.
 52. The apparatus according to claim 45, whereinsaid reagent dispenser is selectively moveable relative to the reagentsupport.
 53. An apparatus according to claim 45, wherein said reagentdispenser comprises at least one robotic dispensing pipette.
 54. Anapparatus according to claim 46, wherein said robotic dispensingpipette(s) is/are vertically moveable between an operating position anda transport position.
 55. An apparatus according to claim 45, whereinsaid reagent dispenser comprises two spaced robotic dispensing pipettes.56. An apparatus according to claim 45, wherein the nozzle supportcarries at least one vortex air jet or jet drain.
 57. An apparatusaccording to claim 45, wherein the nozzle support carries at least oneapplicator for applying a cover fluid to the slide.
 58. (canceled) 59.An apparatus according to claim 45, wherein the nozzle support isrotatably moveable plus or minus approximately 180° from a homeposition.
 60. An apparatus according to claim 45, wherein said reagentdispenser is rotatably moveable plus or minus approximately 185° from ahome position.
 61. An apparatus according to claim 45, and furthercomprising a slide bar code reader carried on the nozzle support and/orcarried with the reagent dispenser.
 62. An apparatus according to claim45, and further comprising a processor for controlling movement of thenozzle support, reagent support and the reagent dispenser.
 63. Anapparatus according to claim 45, and further comprising a dispenser washstation mounted on the nozzle support.
 64. An apparatus according toclaim 62, and comprising two dispenser wash stations mounted spaced fromone another on the nozzle support.
 65. (canceled)